ACE (Angiotensin Converting Enzyme) efficient degrading flora cultivated based on emergent aquatic plant root system as well as method and application thereof
By screening and accumulating ACE degradation bacteria in the roots of the water plant, the problem of ACE pollution in surface water was solved, and efficient degradation of ACE in the wetland system was achieved, with a high degradation rate and no secondary pollution.
Patent Information
- Application Number
- CN202510260511.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-05-30
AI Technical Summary
There is high concentration of persistent ACE pollution in the surface water environment, which leads to ecological security problems and human health hazards, and it is difficult to effectively remove existing sewage treatment processes.
By screening and accumulating dominant degradation bacteria in the root system of the water plant, efficient ACE degradation bacteria can be obtained, and efficient ACE degradation is quickly achieved through direct investment in the wetland system.
It has achieved efficient removal of ACE in the wetland system, with a degradation rate of 0.39h-1, and can completely degrade 100mg/L of ACE within 24h. The degradation products are inorganic sulfamic acid, water and carbon dioxide.
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Figure CN120060111A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of environmental ecological restoration, and specifically relates to an ACE highly efficient degradation bacterial community based on the cultivation of emergent plant roots, its method and application. Background Art
[0002] Acesulfame (ACE) is a synthetic additive widely used in food, beverages, pharmaceuticals and animal feeds. Because of its extremely high sweetness, up to 200 times that of sucrose, it is one of the most widely used artificial sweeteners in the world. ACE is not absorbed by the human body or animals and almost all enters the sewage system through excretion. ACE has strong chemical and biological stability, and it is difficult for sewage treatment processes to effectively remove it. Therefore, most of the ACE penetrates the sewage treatment system and enters the water environment. The highest concentration of ACE in the influent of the Swiss sewage treatment system can reach 61.2 μg / L, and it reaches 7.0 μg / L in surface water; the average value of ACE in the Rhine River and the Main River in Germany is higher than 36.0 μg / L; the highest concentration of ACE detected in the water source of Hangzhou City, China is 22.9 μg / L, and the detection rate can reach 72.7%. The high-concentration persistent ACE pollution in the surface water environment may cause ecological safety problems and may also affect the water quality of water sources, thereby posing a hazard to human health. ACE has a high solubility and is difficult to degrade in the environment. Under the conditions of extensive use and continuous discharge, ACE will continue to accumulate in the water environment. Strengthening the ecological restoration function is the key to effectively solving the pollution of trace persistent artificial sweeteners in the water environment, and artificial or natural wetlands are effective ways for the ecological restoration of the water environment.
[0003] The present invention combines phytoremediation and microbial technology, screens dominant degrading bacteria from the roots of emergent plants, and the obtained highly efficient bacteria can rapidly and simply achieve the highly efficient degradation of ACE in the wetland system by direct injection. Summary of the Invention
[0004] Based on the governance requirements of trace persistent organic pollution in the water environment, the purpose of the present invention is to provide a method for domesticating and screening ACE highly efficient degrading bacteria based on the microorganisms in the roots of emergent plants, and strengthening the treatment ability of wetlands for ACE, so as to solve the problem of trace organic pollution in the water environment through ecological restoration.
[0005] The technical solution adopted by the present invention is specifically as follows:
[0006] A method for cultivating an ACE highly efficient degradation bacterial community based on the roots of emergent plants, comprising the following steps:
[0007] 1) Domestication: Plant emergent plants into the wetland system after light-shielding treatment, add the domestication target solution to the wetland system, sample the wetland system at regular intervals to measure the content of acesulfame potassium (ACE), and replace the domestication target solution. When the ACE removal rate in the sampled domestication target solution exceeds 90%, it indicates that sufficient ACE-degrading bacteria communities are generated in the plant roots. The wetland system can be a simulated wetland system or a real wetland system;
[0008] 2) Isolation: Isolate the initial bacterial solution of the ACE-degrading bacteria communities from the plant roots, and inoculate the initial bacterial solution into a liquid medium;
[0009] 3) Cultivation: Under constant temperature conditions, cultivate the inoculated liquid medium until the logarithmic growth phase, and then streak the bacterial solution at the logarithmic growth phase on an LB solid medium. After culturing for a period of time, colonies are obtained;
[0010] 4) Screening: Pick colonies and inoculate them into a liquid medium to verify whether the colonies have the ability to degrade ACE, and retain the colonies with the ability to degrade ACE;
[0011] 5) Purification: Repeat steps 3) and 4) for the colonies with the ability to degrade ACE in step 4) until the colonies with the ability to degrade ACE reach the required degradation rate, that is, obtain the highly efficient ACE-degrading bacteria communities;
[0012] Among them, the liquid medium is DMSZ 462 liquid medium with ACE as the sole carbon source.
[0013] Further, in step 1), the emergent plants are one or more of Scirpus validus and Phyllostachys heteroclada Oliver. The emergent plants are supplemented with lighting using a full-spectrum plant growth lamp, the light intensity is 5000 - 7000 lx, and the light-dark ratio is 16 h:8 h.
[0014] Further, in step 1), the wetland system includes: water and a substrate. The substrate uses gravel to cover the roots of the emergent plants.
[0015] Further, in step 1), the domestication target solution is a nutrient solution containing ACE. The liquid level of the nutrient solution is at least 3 - 8 cm higher than the gravel, and the domestication target solution is replaced every 1 - 3 days; preferably, the liquid level of the nutrient solution is at least 5 cm higher than the gravel, and the domestication target solution is replaced every 2 days.
[0016] Further, in step 2), the isolation is specifically: Take the plant roots and ultrasonicate them at 50 Hz, and discard the supernatant after centrifugation.
[0017] Furthermore, in step 3), the constant temperature is 25-28°C, and the culturing period is specifically 36-60 hours; preferably, the constant temperature is 28°C, and the culturing period is specifically 48 hours.
[0018] Furthermore, in step 5), the colonies with ACE degradation ability achieve the required degradation rate specifically: the colonies with ACE degradation ability can completely degrade ACE in the DMSZ 462 liquid culture medium within 24 hours.
[0019] A highly efficient ACE-degrading bacterial community cultivated based on the root system of emergent plants is obtained by any of the methods described, wherein the highly efficient ACE-degrading bacterial community comprises: Shinella, Sphingobacterium and Taibaiella; the highly efficient ACE-degrading bacterial community is suitable for the degradation of ACE in an environment of pH 6-9 and temperature of 20-35°C; preferably, the highly efficient ACE-degrading bacterial community is suitable for the degradation of ACE in an environment of pH 7 and temperature of 35°C.
[0020] The application of the ACE efficient degrading bacteria in wetland restoration is to obtain the ACE degrading bacteria by directly planting emergent plants in the wetland for domestication or directly adding the ACE efficient degrading bacteria and fixing them in the wetland to enhance the ACE degradation capacity of the wetland.
[0021] An ACE-degrading bacterial agent comprises the ACE-efficient degrading bacterial flora.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] 1. The ACE-efficient degrading bacterial community of the present invention can be attached to the wetland matrix and can efficiently remove ACE without relying on symbiotic emergent plants;
[0024] Second, the ACE efficient degradation bacterial flora of the present invention has a highly efficient degradation ability for ACE, and can completely degrade ACE with an initial concentration of 100 mg / L within 24 hours, with a degradation rate of 0.39 h-1;
[0025] 3. The degradation products of ACE by the high-efficiency ACE-degrading bacteria are inorganic aminosulfonic acid, water and carbon dioxide, which can completely mineralize ACE;
[0026] 4. The ACE-efficient degrading bacteria have strong environmental adaptability and can degrade ACE in the range of pH 6-9 and temperature 20℃-35℃;
[0027] 5. The ability of wetlands to remove ACE can be enhanced quickly and simply by directly adding and fixing ACE-degrading bacteria or ACE-efficient degrading bacteria obtained by domesticating emergent plants planted in wetlands. Brief Description of the Drawings
[0028] Figure 1 It is a time graph of domesticating an ACE highly efficient degrading bacterial community based on emergent plants in an embodiment of the present invention;
[0029] Figure 2 It is a colony morphology graph of an ACE highly efficient degrading bacterial community in an embodiment of the present invention;
[0030] Figure 3 It is the microscopic morphology of an ACE highly efficient degrading bacterial community in an embodiment of the present invention;
[0031] Figure 4 It is a species community Bar graph of an ACE highly efficient degrading bacterial community in an embodiment of the present invention;
[0032] Figure 5 It is a species community Circos graph of an ACE highly efficient degrading bacterial community in an embodiment of the present invention;
[0033] Figure 6 It is a phylogenetic evolution tree of an ACE highly efficient degrading bacterial community in an embodiment of the present invention;
[0034] Figure 7 It is the wetland removal effect of an ACE highly efficient degrading bacterial community - substrate in an embodiment of the present invention;
[0035] Figure 8 It is a comparison graph of an ACE highly efficient degrading bacterial community and plants on the degradation of ACE in a wetland system in an embodiment of the present invention;
[0036] Figure 9 It is a graph of the degradation rate and degradation rate K of an ACE highly efficient degrading bacterial community on ACE in an embodiment of the present invention;
[0037] Figure 10 It is an ACE degradation curve graph of an ACE highly efficient degrading bacterial community under different pH conditions in an embodiment of the present invention;
[0038] Figure 11 It is an ACE degradation curve graph of an ACE highly efficient degrading bacterial community under different temperature conditions in an embodiment of the present invention. Detailed Embodiments
[0039] The technical solutions of the present invention will be further specifically described below through specific implementation cases and in combination with the drawings. The actual cases are only used for the description of the present invention, rather than limiting conditions of the present invention.
[0040] In the following embodiments, the composition of the Hoagland growth nutrient solution used in the experiment includes:
[0041] Solution A: Ca(NO 3 )2 · 4H 2 O 945 mg / L, KNO 3 506 mg / L, NH 4 NO 3 80 mg / L, NH 4 Cl 15 mg / L;
[0042] Solution B: KH 2 PO 4 136 mg / L, MgSO 4 493 mg / L;
[0043] Iron salt: FeSO 4 · 7H 2 O 13.9 mg / L, EDTA-2Na 18.7 mg / L;
[0044] Trace elements: KI 0.83 mg / L, H 3 BO 3 6.2 mg / L, MnSO 4 22.3 mg / L, Na 2 MoO 4 0.25 mg / L, CuSO 4 0.025 mg / L, CoCl 2 0.025 mg / L, and the rest is sterile water.
[0045] Among them, Solution A and Solution B need to be prepared separately to avoid precipitation. Before use, mix the above solutions and adjust the pH to 6.0 to ensure efficient absorption of nutrients by plants. In addition, the iron salt and trace element solutions should also be prepared separately and stored separately under low temperature and dark conditions. After the iron salt stock solution is prepared, the pH needs to be adjusted to 5.5 to prevent precipitation and improve stability.
[0046] The composition of the DMSZ 462 liquid medium used in the experiment includes:
[0047] KH 2 PO 4 1520 mg / L, Na 2 HPO 4 2440 mg / L, (NH 4 ) 2 SO 4 500 mg / L, MgSO 4 · 7H 2 O 200 mg / L, CaCl 2 · 2H 2 O 50 mg / L, SL-4 10 mL / L, vitamin solution 2.5 mL / L, and the rest is sterile water;
[0048] Among them, the vitamin solution includes: 10 mg / L of p-aminobenzoic acid, 2 mg / L of biotin, 20 mg / L of VB 3 20, 10 mg / L of calcium pantothenate, 5 mg / L of VB 1 hydrochloride, 50 mg / L of VB 6 50, 20 mg / L of VB 12 20; The SL-6 solution includes: 100 mg / L of ZnSO 4 ·7H 2 O, 30 mg / L of MnCl 2 ·4H 2 O,
[0049] H 3 BO 3 3, 200 mg / L of CoCl 2 ·6H 2 O, 10 mg / L of CuCl 2 ·2H 2 O, 20 mg / L of NiCl 2 ·6H 2 O, 30 mg / L of Na 2 MoO 4 ·2H 2 O; The SL-4 solution includes: 500 mg / L of EDTA-2Na, 200 mg / L of FeSO 4 ·7H 2 O, 100 mL / L of SL-6.
[0050] The composition of the LB solid medium used in the experiment is: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, 15 g / L of agar, and the rest is sterile water. It needs to be sterilized in an autoclave (sterilization pressure is 103.4 kPa, temperature is 121.3 °C, and maintained for 30 min).
[0051] Example 1:
[0052] Domestication and screening of the ACE highly efficient degradation bacterial community based on emergent plants, namely highly efficient bacteria
[0053] Select Scirpus validus, Schizostachyum diffusum, and Acorus tatarinowii with similar size and weight, well-developed root systems, and good growth states. Transplant all the plants into a simulated wetland system with gravel as the substrate. The roots of all the plants are covered by gravel, and the gravel needs to be sterilized in a high-pressure steam sterilizer before use. The gravel above and below the water surface is covered with black plastic bags to avoid light (to prevent the growth of algae and affect the experimental results). Then, add the acclimation target solution, which is a 10% Hoagland solution containing 20 mg / L ACE. Among them, the 10% Hoagland solution is used as the nutrient solution. The liquid level of the acclimation target solution is 5 cm higher than the gravel. Replace the acclimation target solution every 2 days. Take samples of the replaced acclimation target solution. After filtering the samples through a 0.22 μm filter membrane, measure the ACE concentration and calculate the ACE removal rate. When the ACE removal rate exceeds 90%, it represents that ACE-degrading bacteria communities are generated in the plant roots. As Figure 1 shown, Scirpus validus and Schizostachyum diffusum began to degrade ACE after 8 days of acclimation and could completely degrade ACE after about 14 days, while the removal effect of Acorus tatarinowii on ACE was not obvious. It can be seen that not any emergent plant can be used for ACE acclimation. During the experiment, the laboratory temperature was 20°C - 25°C, the pH was 7, and a full-spectrum plant growth lamp was used to supplement lighting for the plants. The light intensity was 6000 lx, and the light-dark ratio was 16 h:8 h.
[0054] Example 2:
[0055] Isolation, purification, and identification of ACE-degrading bacteria communities
[0056] (1) Isolation and purification:
[0057] ① Take out 5 g of root tissue from the plant roots in Example 1, add 200 mL of sterile water, and ultrasonically oscillate at 50 Hz for 5 min to make the microorganisms attached to the roots fall off into the water. Put the obtained suspension into a centrifuge at 4000 r / min for 15 min, discard the supernatant, add 50 mL of sterile water again, and shake well to obtain the initial bacterial solution of the ACE-degrading bacteria community. Inoculate it into a DMSZ 462 liquid medium with 100 mg / L ACE as the sole carbon source;
[0058] ② Incubate the inoculated liquid medium at a constant temperature of 28°C until the logarithmic growth phase; streak the bacterial solution at the logarithmic growth phase on an LB solid medium and incubate it at a constant temperature of 28°C for 48 h to obtain colonies;
[0059] ③ Pick colonies with different colors and morphologies, inoculate them again into the DMSZ 462 liquid medium, verify whether the colonies have the ability to degrade ACE, and retain the colonies with the ability to degrade ACE;
[0060] ④ Repeat steps ② and ③ for the colonies with ACE degradation ability in step ③, until the colonies with ACE degradation ability can completely degrade ACE in the DMSZ 462 liquid culture medium within 24 hours, that is, obtain a bacterial colony that can efficiently degrade ACE. In step ③, verify whether the colonies have ACE degradation ability by inoculating the colonies into the liquid culture medium at 28°C and culturing them at a constant temperature until the logarithmic growth phase, and confirm whether there is ACE degradation ability and its degradation rate by measuring the ACE content in the liquid culture medium at this time. Figure 2 As shown in the figure, the colonies grown in LB medium are yellow, bulging in the middle, relatively smooth, with complete edges, shiny and sticky. Figure 3 As shown, there are three types of strains with different morphologies in the degrading bacteria, namely thick rod-shaped, thin rod-shaped, and paramecium-shaped, without flagella and spores.
[0061] (2) Identification: High-throughput sequencing technology was used to identify the ACE-efficient degrading bacteria. Figure 4 , 5 and 6. The main dominant genera were Shinella (24.61%), Sphingobacterium (24.46%), and Taibaiella (8.66%).
[0062] Embodiment 3:
[0063] ACE efficient degradation bacteria-matrix wetland removal of ACE
[0064] According to the acclimation method of Example 1, after the simulated wetland system produced a stable degradation effect on ACE, the plants water plantain and water bamboo were removed, and the degradation effect of the remaining matrix on ACE was still above 90%. Figure 7 As shown, it is shown that the ACE efficient degradation bacterial community of the present invention can be attached to the wetland matrix and can efficiently remove ACE without relying on symbiotic emergent plants.
[0065] Embodiment 4:
[0066] Performance of high-efficiency bacteria in wetland systems
[0067] According to the domestication method of Example 1, a wetland system containing water plantain and water bamboo was domesticated, and the ACE efficient degradation bacteria described in Example 2 were added to the wetland system containing only gravel to verify the effect of the ACE efficient degradation bacteria on the degradation of ACE in the wetland system. The experimental results show that ( Figure 8As shown, the conventional domestication method takes 14 - 16 days to mature the wetland system and can completely remove ACE; while directly adding the ACE - efficient degradation bacterial community only takes 2 - 4 days to enable the wetland system to acquire the ability to efficiently remove ACE, and can maintain a high degradation rate all the time, indicating that this ACE - efficient degradation bacterial community can firmly adsorb on the gravel in the wetland system (immobilized through physical, chemical, and biological actions in the gravel), and quickly and simply strengthen the function of the wetland system to remove ACE.
[0068] Example 5:
[0069] Degradation of ACE by the efficient degradation bacterial community
[0070] The bacterial community that efficiently degrades ACE described in Example 2 was inoculated into 200 mL of DMSZ 462 liquid medium containing 100 mg / L of ACE, with a pH of 7. It was placed in an incubator at a constant temperature of 28°C, and samples were taken every 4 hours to detect the remaining ACE concentration. The degradation rate and degradation rate constant K of the efficient degradation bacterial community for ACE are as Figure 9 shown. The test results indicate that the process of the degradation bacterial community degrading ACE can be divided into two stages: a lag phase and an active phase. The degradation rate constant K in the lag phase 1 is 0.0048 h -1 (R 2 = 0.869), and the degradation rate constant K in the active phase 2 is 0.390 h -1 (R 2 = 1). It can completely degrade 100 mg / L of ACE within 24 h, demonstrating high - efficient degradation ability.
[0071] Example 6:
[0072] According to the method of the ACE - efficient degradation bacterial community degradation ability test in Example 5, with an ACE concentration of 100 mg / L, the pH of the DMSZ 462 liquid medium was adjusted to 5, 6.5, 7, 9, and 11, and the ACE degradation degree was detected. The test results are as Figure 10 shown, indicating that the optimal pH of the ACE - efficient degradation bacterial community is 7, and it can completely degrade ACE within 24 h.
[0073] Example 7:
[0074] According to the method of the ACE - efficient degradation bacterial community degradation ability test in Example 5, with an ACE concentration of 100 mg / L, the culture temperature was adjusted to 5°C, 15°C, 25°C, and 35°C, and the ACE degradation degree was detected. The test results are as Figure 11 shown, indicating that the optimal growth temperature of the efficient degradation bacterial community is 35°C, and it can completely degrade ACE within 24 h.
[0075] The test results according to Example 6 and Example 7 show that the highly efficient degradation bacterial community has strong environmental adaptability and can have a good degradation effect on ACE under environmental conditions with a pH of 6 - 11 and a temperature of 20°C - 35°C. The optimal degradation conditions are a pH of 7 and a temperature of 35°C.
[0076] In summary, the present invention provides a method for domesticating and selecting a highly efficient ACE-degrading bacterial community using emergent plants and strengthening the removal of trace persistent ACE in wetlands through the fixation of microorganisms and substrates. This method is simple, easy to implement, green, safe, without secondary pollution, and low in cost, and can provide a technical method for the ecological restoration of trace persistent organic pollutants in the water environment.
[0077] The above-described embodiments are only preferred solutions of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions described in the claims.
Claims
1. A method for cultivating ACE efficient degradation bacteria based on the root system of emergent plants, characterized in that: The following steps are involved: 1) Acclimation: Plant emergent plants in a wetland system that has been protected from light, add an acclimation target solution to the wetland system, sample the wetland system at regular intervals to determine the acesulfame potassium (ACE) content, and replace the acclimation target solution. When the ACE removal rate in the sampled acclimation target solution exceeds 90%, it means that the plant root system produces a sufficient amount of ACE-degrading bacteria; 2) Isolation: isolating an initial bacterial liquid of ACE-degrading bacteria from the plant root system, and inoculating the initial bacterial liquid into a liquid culture medium; 3) Cultivation: Under constant temperature conditions, the inoculated liquid culture medium is cultured to the logarithmic growth phase, and then the bacterial liquid that has reached the logarithmic growth phase is streaked on the LB solid culture medium, and colonies are obtained after culturing for a period of time; 4) Screening: Pick colonies and inoculate them into liquid culture medium to verify whether the colonies have the ability to degrade ACE, and retain colonies that have the ability to degrade ACE; 5) Purification: Repeat steps 3) and 4) for the colonies with ACE degradation ability in step 4) until the colonies with ACE degradation ability reach the required degradation rate, thus obtaining ACE efficient degradation bacterial population; Wherein, the liquid culture medium is DMSZ 462 liquid culture medium using ACE as the sole carbon source.
2. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 1, characterized in that: In step 1), the emergent plants are one or more of water plantain and water bamboo, and the emergent plants are supplemented with lighting using a full-spectrum plant growth lamp with a light intensity of 5000-7000 lx and a light-dark ratio of 16h:8h.
3. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 1, characterized in that: In step 1), the wetland system comprises: water and a substrate, wherein the substrate is gravel, and the roots of the emergent plants are covered by the gravel.
4. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 3, characterized in that: In step 1), the target acclimation solution is a nutrient solution containing ACE, the liquid level of the nutrient solution is at least 3-8 cm higher than the gravel, and the target acclimation solution is replaced every 1-3 days.
5. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 1, characterized in that: In step 2), the separation is specifically as follows: taking the plant roots and ultrasonicating them at 50 Hz, and discarding the supernatant after centrifugation.
6. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 1, characterized in that: In step 3), the constant temperature is 25-28° C., and the culturing period is specifically 36-60 hours.
7. The method for cultivating ACE efficient degradation bacteria based on emergent plant root system according to claim 1, characterized in that: In step 5), the colonies with ACE degradation ability reach the required degradation rate specifically: the colonies with ACE degradation ability can completely degrade ACE in the DMSZ 462 liquid culture medium within 24 hours.
8. An ACE-efficient degradation bacterial community cultivated based on the root system of emergent plants, characterized in that: The method according to any one of claims 1 to 7 is used to obtain the ACE-efficient degrading bacterial community, which includes Shinella, Sphingobacterium and Taibaiella; the efficient degrading bacterial community is suitable for the degradation of ACE in an environment of pH 6-9 and temperature of 20°C-35°C.
9. The use of the ACE efficient degradation bacterial flora in wetland restoration according to claim 8, characterized in that: ACE-degrading bacteria are obtained by directly planting emergent plants in wetlands and domesticating them, or ACE-efficient degrading bacteria are directly added and fixed in wetlands.
10. An ACE-degrading bacterial agent, characterized in that: The method comprises the ACE-efficient degrading bacterial flora as described in claim 8.